Endocine™, N3OA and N3OASq; three mucosal adjuvants that enhance the immune response to nasal influenza vaccination.

Annual outbreaks of seasonal influenza are controlled or prevented through vaccination in many countries. The seasonal vaccines used are either inactivated, currently administered parenterally, or live-attenuated given intranasally. In this study three mucosal adjuvants were examined for the influence on the humoral (mucosal and systemic) and cellular influenza A-specific immune responses induced by a nasally administered vaccine. We investigated in detail how the anionic Endocine™ and the cationic adjuvants N3OA and N3OASq mixed with a split inactivated influenza vaccine induced influenza A-specific immune responses as compared to the vaccine alone after intranasal immunization. The study showed that nasal administration of a split virus vaccine together with Endocine™ or N3OA induced significantly higher humoral and cell-mediated immune responses than the non-adjuvanted vaccine. N3OASq only significantly increased the cell-mediated immune response. Furthermore, nasal administration of the influenza vaccine in combination with any of the adjuvants; Endocine™, N3OA or N3OASq, significantly enhanced the mucosal immunity against influenza HA protein. Thus the addition of these mucosal adjuvants leads to enhanced immunity in the most relevant tissues, the upper respiratory tract and the systemic circulation. Nasal influenza vaccination with an inactivated split vaccine can therefore provide an important mucosal immune response, which is often low or absent after traditional parenteral vaccination.

DNA-Encoded Flagellin Activates Toll-Like Receptor 5 (TLR5), Nod-like Receptor Family CARD Domain-Containing Protein 4 (NRLC4), and Acts as an Epidermal, Systemic, and Mucosal-Adjuvant.

Eliciting effective immune responses using non-living/replicating DNA vaccines is a significant challenge. We have previously shown that ballistic dermal plasmid DNA-encoded flagellin (FliC) promotes humoral as well as cellular immunity to co-delivered antigens. Here, we observe that a plasmid encoding secreted FliC (pFliC(-gly)) produces flagellin capable of activating two innate immune receptors known to detect flagellin; Toll-like Receptor 5 (TLR5) and Nod-like Receptor family CARD domain-containing protein 4 (NRLC4). To test the ability of pFliC(-gly) to act as an adjuvant we immunized mice with plasmid encoding secreted FliC (pFliC(-gly)) and plasmid encoding a model antigen (ovalbumin) by three different immunization routes representative of dermal, systemic, and mucosal tissues. By all three routes we observed increases in antigen-specific antibodies in serum as well as MHC Class I-dependent cellular immune responses when pFliC(-gly) adjuvant was added. Additionally, we were able to induce mucosal antibody responses and Class II-dependent cellular immune responses after mucosal vaccination with pFliC(-gly). Humoral immune responses elicited by heterologus prime-boost immunization with a plasmid encoding HIV-1 from gp160 followed by protein boosting could be enhanced by use of pFliC(-gly). We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity. These observations indicate that plasmid-encoded secreted flagellin can activate multiple innate immune responses and function as an adjuvant to non-living/replicating DNA immunizations. Moreover, the capacity to elicit mucosal immune responses, in addition to dermal and systemic properties, demonstrates the potential of flagellin to be used with vaccines designed to be delivered by various routes.

The Eurocine L3 adjuvants with subunit influenza antigens induce protective immunity in mice after intranasal vaccination.

The immunogenicity and protective efficacy in mice of intranasally (i.n.) administrated influenza subunit antigens together with lipid-based adjuvants (Eurocine) were compared to those of subcutaneous (s.c.) immunisation. Influenza hemagglutination inhibition (HAI) and ELISA IgG titers were similar in the group's vaccinated s.c. and after i.n. vaccination with adjuvants. The virus-specific IgA levels in serum were higher after vaccination i.n. with adjuvant than after s.c immunisation. Virus-specific IgA was measurable in nasal washings only after i.n vaccinations, with and without adjuvants. Thus, i.n. vaccination with the endogenous non-toxic, lipid adjuvants induced equal or stronger antibody responses as compared to s.c. immunisation with the same antigen. We further analysed the protective efficacy against virus challenge in a mouse model. A subunit antigen preparation of the A/New/Caledonia/20/99 strain was used for vaccination of NMRI mice with different combinations of adjuvants. The mice were challenged i.n. with 6.5 tissue culture infectious doses(50) of homologous virus and sacrificed 3 days later. Since the virus is not lethal in mice, the protective efficacy was measured by quantitative, real-time PCR on pulmonary tissue, obtained at autopsy. The mice treated with only adjuvant and the group of naïve mice clearly had the highest mean viral RNA copy numbers (19.200 and 11.000, respectively). All vaccinated groups had significantly lower copy numbers, especially the mice that received the L3A i.n. (-median 120; i.n. L3B-median 2.200; and non-adjuvanted s.c. vaccination-median 2.600). Our findings prompt further investigations of the effect of the formulations in ferrets, monkeys and man.

Intranasal immunization of young mice with a multigene HIV-1 vaccine in combination with the N3 adjuvant induces mucosal and systemic immune responses.

One of the major challenges for the development of an HIV vaccine is to induce potent virus-specific immune responses at the mucosal surfaces where transmission of virus occurs. Intranasal delivery of classical vaccines has been shown to induce good mucosal antibody responses, but so far for genetic vaccines the success has been limited. This study shows that young individuals are sensitive to nasal immunization with a genetic vaccine delivered in a formulation of a lipid adjuvant, the Eurocine N3. Intranasal delivery of a multiclade/multigene HIV-1 genetic vaccine gave rise to vaginal and rectal IgA responses as well as systemic humoral and cellular responses. As electroporation might become the preferred means of delivering genetic vaccines for systemic HIV immunity, nasal delivery by droplet formulation in a lipid adjuvant might become a means of priming or boosting the mucosal immunity.

Safety and immunogenicity, after nasal application of HIV-1 DNA gagp37 plasmid vaccine in young mice.

BACKGROUND: There is a need for safe and potent adjuvants capable of delivering vaccine candidates over the mucosal barrier, with good capacity to stimulate both mucosal and systemic cell-mediated and humoral immunity. An adjuvant aimed for intranasal delivery should preferably deliver the antigen and minimize the transfer into the close proximity of the central nervous system, thus avoiding damage on the olfactory tissues. Advantages with a mucosal delivery route would be to provide mucosal and systemic immunity, requiring lower vaccine doses then when given parentally. The aim of this study was to study if the N3 adjuvant intranasally administered with HIV DNA plasmids would be transferred into the olfactory tissues and cause local inflammation and tissue damage. RESULTS: The N3 adjuvant alone and when combined with HIV-1 DNA gag plasmid and delivered intranasally did not cause detectable damage to the nasal epithelium or the olfactory epithelium or bulb over a period of 3 days after delivery. The intranasal administration of HIV-1 gagp37 DNA induced both a humoral and a cell-mediated immunity against the gag antigen. Significantly higher HIV-1-specific humoral, but not cell-mediated immune responses were seen in DNA/N3-immunized mice in comparison with HIV-1 DNA/saline-immunized animals. CONCLUSIONS: A safe and convenient intranasal mode of HIV-1 DNA plasmid and adjuvant delivery was shown not to interfere with the tissues in close proximity to the central nervous system. The N3 adjuvant combined with HIV-1 plasmids enhances the HIV-1-specific immunogenicity and merits to be clinically tested.

A novel DNA adjuvant, N3, enhances mucosal and systemic immune responses induced by HIV-1 DNA and peptide immunizations.

AIMS: The study was designed to evaluate a novel cationic lipid DNA adjuvant (N3) and its function for HIV-1gp160/rev DNA plasmid delivered intranasally. The primary N3/HIV-DNA plasmid immunizations were boosted intranasally with a gp41 peptide in a anionic L3 adjuvant. This novel prime-boost strategy of mucosal immunization provided a broad HIV-1 envelope specific immunity, and recognition of viruses of subtypes A, B and C. CONCLUSIONS: Intranasal N3-adjuvanted gp160/rev DNA prime followed by one L3-peptide boosting immunization, induced broadly neutralizing antibodies against HIV-1 in the mucosa and systemically. The needle-free intranasal prime-boost strategy using two different adjuvant formulations reduced significantly the dose of DNA needed.

Phosphorus homeostasis in dairy cows with abomasal displacement or abomasal volvulus.

Abnormal phosphorus homeostasis occurs in dairy cows with an abomasal displacement or volvulus. The goal of this study was to identify potential mechanisms for hypophosphatemia and hyperphosphatemia in cows with a left displaced abomasum (LDA), right displaced abomasum (RDA), or abomasal volvulus (AV). Accordingly, the results of preoperative clinicopathologic analyses for 1,368 dairy cows with an LDA (n = 1,189), RDA, or AV (n = 179) (data set 1) and for 44 cows with an AV (data set 2) were retrieved. Laboratory values were compared by Student's t-tests, and correlation and regression analyses were performed. Thirty-four percent of the animals from data set 1 (463/1,368) were hypophosphatemic (serum phosphorus concentration ([Pi]) < 1.4 mmol/L), and 9% (122/1,368) were hyperphosphatemic ([Pi] >2.3 mmol/L). Serum [Pi] was significantly lower (P < .05) in cows with an LDA (1.60 +/- 0.53 mmol/L; mean +/- SD) than in cows with an RDA or AV (1.85 +/- 0.68 mmol/L). For cows with an LDA, [Pi] was correlated with serum urea nitrogen concentration ([SUN]) (r = 0.34) and serum concentration of magnesium ([Mg]) (r = 0.20). For cows with an RDA or AV, linear correlations existed between [Pi] and [SUN] (r = 0.45), [Mg] (r = 0.43), and serum chloride concentration ([Cl]) (r = -0.27). Stepwise logistic regression analysis indicated that low [SUN] and the diagnosis of an LDA had the strongest associations with hypophosphatemia. In cows with hyperphosphatemia, [Pi] was most strongly associated with azotemia. In cows with an AV, the strongest correlations with [Pi] were found for [SUN] and serum creatinine. We conclude that hypophosphatemia in cows with an LDA is primarily due to decreased feed intake. In contrast, hyperphosphatemia in cattle with an RDA or AV appears to result from dehydration and decreased renal blood flow.

Intranasal HIV-1-gp160-DNA/gp41 peptide prime-boost immunization regimen in mice results in long-term HIV-1 neutralizing humoral mucosal and systemic immunity.

An intranasal DNA vaccine prime followed by a gp41 peptide booster immunization was compared with gp41 peptide and control immunizations. Serum HIV-1-specific IgG and IgA as well as IgA in feces and vaginal and lung secretions were detected after immunizations. Long-term humoral immunity was studied for up to 12 mo after the booster immunization by testing the presence of HIV-1 gp41- and CCR5-specific Abs and IgG/IgA-secreting B lymphocytes in spleen and regional lymph nodes in immunized mice. A long-term IgA-specific response in the intestines, vagina, and lungs was obtained in addition to a systemic immune response. Mice immunized only with gp41 peptides and L3 adjuvant developed a long-term gp41-specific serum IgG response systemically, although over a shorter period (1-9 mo), and long-term mucosal gp41-specific IgA immunity. HIV-1-neutralizing serum Abs were induced that were still present 12 mo after booster immunization. HIV-1 SF2-neutralizing fecal and lung IgA was detectable only in the DNA-primed mouse groups. Intranasal DNA prime followed by one peptide/L3 adjuvant booster immunization, but not a peptide prime followed by a DNA booster, was able to induce B cell memory and HIV-1-neutralizing Abs for at least half of a mouse's life span.

Mycobacterium tuberculosis arabinomannan-protein conjugates protect against tuberculosis.

Lipoarabinomannan (LAM) is a major structural surface component of mycobacteria. Arabinomannan (AM) oligosaccharides derived from LAM of Mycobacterium tuberculosis H37Rv were isolated and covalently conjugated to tetanus toxoid (TT) or to short-term culture filtrate proteins (antigen 85B (Ag85B) or a 75kDa protein) from M. tuberculosis strain Harlingen. The different AM oligosaccharide (AMOs)-protein conjugate vaccine candidates proved to be highly immunogenic, inducing boosterable IgG responses against the AMOs portion of the conjugates in rabbits and guinea-pigs. Proliferation of T-cells from C57BL/6 mice immunized with the conjugates was seen upon in vitro stimulation with PPD. In C57BL/6 mice subcutaneous immunization with the AMOs-antigen 85B conjugate in alum provided significant protection compared to sham (alum only) immunized mice (P < 0.021) as estimated by long term survival against intravenous challenge with 10(5) M. tuberculosis H37Rv. Subcutaneous immunization followed by nasal boost with an AMOs-TT conjugate in Eurocine L3 adjuvant provided high (P < 0.025) protection as determined by long term survival after intranasal challenge with 10(5) virulent M. tuberculosis strain Harlingen. This level of protection was comparable to that obtained with the conventional live attenuated BCG vaccine. In guinea-pigs, immunization with AMOs-Ag85B in Eurocine L3 adjuvant followed by aerogenic challenge with M. tuberculosis H37Rv resulted in increased survival and reduced pathology in lungs and spleens relative to non-immunized animals.

Immunogenicity and protective efficacy of a formalin-inactivated rotavirus vaccine combined with lipid adjuvants.

The immunogenicity and protective efficacy of inactivated rotavirus vaccine administered intramuscularly with lipid adjuvants; MPL (monophosphoryl lipid A from Salmonella minnesota) or L3 (monooleate/lauric acid) was evaluated in an infant mouse model. Purified and formalin-inactivated rhesus rotavirus (I-RRV) combined with one of the adjuvants were administered to female balb/c mice at 0, 4 and 8 weeks. High serum IgG antibody titers developed in all vaccinated groups; I-RRV (GMT 45524+/-9819), I-RRV-MPL (GMT 190637+/-64250) and I-RRV-L3 (GMT 126266+/-27553). The formalin-inactivation procedure preserved neutralizing epitopes and elicited high neutralizing antibody titers; I-RRV (GMT 43053 S.E.M.+/-4189), I-RRV-MPL (GMT 66398 S.E.M.+/-20202) and I-RRV-L3 (GMT 60887 S.E.M.+/-10750). All offsprings to immunized dams were protected against clinical diarrhea upon oral challenge with RRV. The IgG1/IgG2a ratio was in all immunized groups approximately 1 suggesting development of a balanced Th1/Th2 response.

Mycobacterial antigens exacerbate disease manifestations in Mycobacterium tuberculosis-infected mice.

To control tuberculosis worldwide, the burden of adult pulmonary disease must be reduced. Although widely used, Mycobacterium bovis BCG vaccination given at birth does not protect against adult pulmonary disease. Therefore, postexposure vaccination of adults with mycobacterial antigens is being considered. We examined the effect of various mycobacterial antigens on mice with prior M. tuberculosis infection. Subcutaneous administration of live or heat-treated BCG with or without lipid adjuvants to infected mice induced increased antigen-specific T-cell proliferation but did not reduce the bacterial load in the lungs and caused larger lung granulomas. Similarly, additional mycobacterial antigen delivered directly to the lungs by aerosol infection with viable M. tuberculosis mixed with heat-killed Mycobacterium tuberculosis (1:1) also did not reduce the bacillary load but caused increased expression of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6), which was associated with larger granulomas in the lungs. When M. tuberculosis-infected mice were treated with recombinant BCG that secreted cytokines shown to reduce disease in a preinfection vaccine model, the BCG secreting TNF-alpha, and to a lesser extent, IL-2 and gamma interferon (IFN-gamma), caused a significant increase in granuloma size in the lungs. Moreover, treatment of M. tuberculosis-infected mice with recombinant murine TNF-alpha resulted in increased inflammation in the lungs and accelerated mortality without affecting the bacillary load. Taken together, these studies suggest that administration of mycobacterial antigens to mice with prior M. tuberculosis infection leads to immune activation that may exacerbate lung pathology via TNF-alpha-induced inflammation without reducing the bacillary load.